Este trabalho foi financiado pelo fundo nacional de ciência para ilustres jovens estudiosos (81625002), Nacional Natural Science Foundation da China (81470389, 81270282, 81601238), programa de Shanghai acadêmica líder de pesquisa (18XD1402400), Shanghai Municipal Apoio educação Comissão Gaofeng medicina clínica Grant (20152209), centro de desenvolvimento do Hospital de Shenkang de Shanghai (16CR3034A), Universidade do Tong de Shanghai Jiao (YG2013MS42), Shanghai Jiao Tong University School of Medicine (15ZH1003 e 14XJ10019), Programa de navegação de Shanghai (18YF1413000) e programa de pós-graduação inovação da faculdade de medicina de Bengbu (Byycx1722). Agradecemos sua ajuda anterior Dr. Erhe Gao em nosso laboratório.

Todas as experiências em animais foram realizadas de acordo com os institutos nacionais de saúde orientações sobre o uso de animais de laboratório e foram aprovadas pelo Comitê de ética Animal do Instituto. Camundongos C57BL/6J machos (8-10 semanas de idade) foram usados para todos os experimentos. Os ratos foram alojados em condições de livre de patógeno a 24 ° C ± 4 ° C, sob um ciclo claro/escuro de 12-h, com livre acesso à água e comida.
1. preparação da solução de adenovírus
<ol><li>Após a chegada da solução purificada adenovírus, conservar a solução em um freezer-80 ° C. Nota: A injeção de adenovírus foi realizada em ratos viáveis. Para garantir a esterilidade do vetor adenovírus, o vírus adenoide (codificação do luciferase, VDR ou shRNA alvo VDR) foram preparadas e purificado comercialmente9,10.</li>
	<li>No dia da operação, retire a solução purificada adenovírus (3 x 1010 placa formando unidades [pfu] /mL) do congelador a-80 ° C e descongelar a solução do vetor de adenovírus no gelo.</li>
	<li>Veste uma máscara, luvas estéreis e vestido de estéril.</li>
	<li>Preparar um 50 µ l seringa Hamilton que foi esterilizada no dia antes da operação. Nota: Para a esterilização, a seringa Hamilton foi envolto em gaze e colocada em um esterilizador de pressão de alta temperatura/alta. O modo de "Esterilização para o sólido" foi selecionado e esterilização foi iniciada pressionando o botão "start".</li>
	<li>Após o descongelamento completo da solução purificada adenovírus, pulverizar o tubo contendo solução de adenovírus com 75% de etanol e mover a solução de adenovírus em uma capa de fluxo laminar estéril.</li>
	<li>Em uma capa de fluxo laminar estéril, Aspire solução de adenovírus 50 µ l utilizando a seringa Hamilton sem uma agulha, seguida de penhora de uma agulha 30G à seringa Hamilton.</li>
	<li>Segurar a seringa com a agulha de 30g para cima e empurre suavemente o êmbolo da seringa até que a agulha é preenchida com solução de adenovírus (confirmada pelo aparecimento das primeiras gotas de solução de ponta da agulha). Nota: Demora aproximadamente 45 µ l solução para preencher a agulha 30G, então agora quase nenhuma solução é visível na seringa.</li>
	<li>Use a seringa preparada acima para Aspire cuidadosamente com outra solução de adenovírus 30 µ l e coloque a seringa vagamente tampada no gelo para uso subsequente.</li>
</ol>2. anestesia e preparação operativa
<ol><li>Adicionar 20 mL isoflurano em um vaporizador de isoflurano e conectar o vaporizador de isoflurano para um tanque de oxigênio.</li>
	<li>Abra a válvula e deixe o oxigênio fluxo do tanque de oxigênio para o vaporizador de isoflurano. Manter a taxa de fluxo de oxigênio a 2 L/min, através de um monitor de fluxo de oxigênio no vaporizador de isoflurano.</li>
	<li>Induzi a anestesia do mouse com 4% de isoflurano em 100% de oxigênio (2 L/min) por 2 min em uma gaiola de plástico ligada ao vaporizador de isoflurano.</li>
	<li>Tirar o mouse anestesiado da jaula de plástico e fixá-lo em uma plataforma de plástico na posição de bruços em uma capa de fluxo laminar estéril e manter a anestesia com isoflurano de 2% em 100% de oxigênio (2 L/min) através de um cone de nariz.</li>
	<li>Confirme a anestesia adequada pela ausência de resposta de pitada de dedo do pé.</li>
	<li>Aplique o creme oftálmico estéril para cada olho para proteger as córneas de secagem.</li>
	<li>Depilar o peito e abdômen superior. Aplicar creme depilatório comercialmente disponível para o site raspado para 1 min. Retire o creme depilatório e restante pele com gazes molhadas.</li>
	<li>Esterilize o local cirúrgico (parte inferior esquerda do tórax) com 3 batas de anti-séptico à base de iodo-clorexidina (por exemplo, entoiodine). Cobrir o local cirúrgico com um pano estéril.</li>
</ol>3. Intramiocárdica injeção de adenovírus no coração de rato
<ol><li>Tirar as luvas e colocar um novo par de luvas estéreis.</li>
	<li>Esterilizar o fórceps, uma pinça hemostática micro-mosquito, um par de tesouras cirúrgicas, e um porta-agulha no dia antes da operação em uma alta temperatura/alta pressão esterilizador (ver nota etapa 1.4).</li>
	<li>Faça uma incisão de 0,5 cm de pele ao longo da linha que liga o xifoide e axila. Sem rodeios, disse o peitoral maior e peitorais menores músculos com fórceps e uma pinça hemostática micro-mosquito.</li>
	<li>Expor os espaços intercostais, retraindo o peitoral maior e peitorais menores músculos. Perfurar e abra o quarto espaço intercostal (ou o mais amplo espaço intercostal por observação) com uma pinça hemostática micro-mosquito.</li>
	<li>Empurre o coração em direção a incisão de exteriorizar o coração com o dedo indicador da mão não dominante, pressionando suavemente contra o lado direito da parede torácica.</li>
	<li>Secure suavemente o coração exteriorizado com o dedo indicador e o polegar da mão não dominante.</li>
	<li>Injete um total de solução de adenovírus 30 µ l do miocárdio do ventrículo esquerdo em três locais (parede ventral, dorsal e lateral do ventrículo esquerdo) através da seringa Hamilton repleto de adenovírus (Figura 1) com a mão dominante.</li>
	<li>Após a conclusão da injeção, coloca imediatamente o coração volta para o espaço intratorácicos.</li>
	<li>Manualmente, evacue o ar no espaço intratorácicos pressionando suavemente a parede torácica para o local de incisão da pele. Nota: Evacuação de ar bem sucedido pode ser evidenciada pelo bater do peitoral maior e peitorais menores músculos causados pelo ar expelido.</li>
	<li>Feche a pele por sutura horizontal com uma sutura de seda 5-0. Nota: O peitoral maior e peitorais menores músculos devem não ser suturados, porque eles só sem rodeios são dissecados e suas estruturas anatômicas estão intactas durante toda a operação.</li>
</ol>4. pós-operatórios
<ol><li>Administre a buprenorfina (3 mg/kg) duas vezes diariamente através de injeção subcutânea para reduzir a dor pós-operatória para as primeiras 48 horas após a operação11.</li>
	<li>Após a operação, imediatamente manter os ratos em uma almofada de calor (37 ° C) até que se recuperou totalmente. Coloque o mouse volta para a jaula, depois que ele se recupere totalmente.</li>
	<li>Esterilizar o usado Hamilton seringa e agulha de seringa 30g num esterilizador alta temperatura/alta pressão (ver nota etapa 1.4). Recolha a agulha de seringa esterilizada 30 G em um recipiente de objectos cortantes.</li>
</ol>5. na Vivo Imaging para medir a expressão Luciferase cardíaca
<ol><li>No dia 5 após a injeção de adenovírus, prepare uma solução stock fresca de luciferina em 15 mg/mL em solução salina de tampão fosfato de Dulbecco (DPBS). Filtre a solução através de um filtro de 0,2 µm.</li>
	<li>Intra peritoneally Injete os ratos acordados com a solução de luciferina a 150 mg/kg de peso corporal.</li>
	<li>Ligue o sistema de imagem.</li>
	<li>Depois automático autoteste do sistema de imagem, selecione o modo de imagem como "Luminescentes" e fazer as seguintes configurações: exposição: Auto; Binning: 8; FStop: 1; Excitação: Bloco; Emissão: aberto.</li>
	<li>Em 10 min após a injeção de luciferina, anestesia os ratos por injeção de hidrato de cloral (300 mg/kg de peso corporal). Confirme a anestesia adequada pela ausência de resposta de pitada de dedo do pé.</li>
	<li>Fixe os ratos anestesiados num palco de imagem aquecida na posição de bruços, com o peito voltado para a câmera.</li>
	<li>Recolher imagens (1 imagem para cada rato) e quantificar a intensidade dos sinais desenhando-se as regiões de idêntica medida circular de interesse (ROI) em torno do peito.</li>
	<li>Após a coleta de imagens, sacrificar os ratos e coletar os tecidos, como mencionado na seção a seguir.</li>
</ol>6. colheita de tecidos
<ol><li>Em pontos diferentes de tempo após a injeção do vírus (figura 1B), anestesiar os ratos com 4% de isoflurano, manter a anestesia com isoflurano 2% através de um cone de nariz e fixá-lo em uma plataforma de plástico na posição de bruços.</li>
	<li>Fazer uma incisão ventral no pescoço anterior. Expor a artéria carótida comum direita, retraindo os músculos omohyoid e esternocleidomastoideo.</li>
	<li>Sacrifica os ratos por cruza a artéria carótida comum direita e drenar o sangue.</li>
	<li>Fazer uma incisão ventral no abdome. Corte as costelas de ambos os lados da caixa torácica, ao longo da linha médio clavicular e transecto o diafragma.</li>
	<li>Expor o coração, levantando o xifoide. Encontrar a aorta ascendente e remova cuidadosamente o tecido adiposo perivascular.</li>
	<li>Canule da aorta e perfundir retrogradely o coração com uma agulha 30G anexada a uma seringa de 1 mL, preenchida com 0,5 mL de solução-tampão de fosfato (PBS) 4 ° C.</li>
	<li>Após a perfusão, excisar o coração e dividi-lo nos ventrículos direito e esquerdos. Excisar o fígado, pulmão e baço.</li>
	<li>Depois de duas lavagens em PBS frio (4 ° C), armazene todos os tecidos coletados separadamente em frascos criogênicos em nitrogênio líquido.</li>
</ol>7. determinação da expressão da proteína
<ol><li>Prepare o tampão de homogeneização adicionando coquetel de inibidor de protease em tampão de Lise comercialmente disponível a uma proporção de 1: 100. Calcule o volume total, com base no número de amostras a serem processadas. Mantenha o buffer de homogeneização preparado a 4 ° C para uso posterior.</li>
	<li>Tire os tecidos do tanque de nitrogênio líquido. Pese o tecido em escalas de alta precisão (cerca de 60 mg para cada pedaço de tecido).</li>
	<li>Transferi o tecido para um novo tubo de 1,5 mL microcentrifuge. Imediatamente adicione 200 µ l homogeneização buffer e pré-resfriado esferas de aço 1,5 mm (4 ° C) no tubo de microcentrifugadora.</li>
	<li>Fixar os tubos microcentrifuga em pré-resfriado (4 ° C) suportes de metal em um tecido automático retificadora e começar a homogeneização, iniciando a máquina com as seguintes configurações: frequência: 70 Hz; tempo: 120 s.</li>
	<li>Após a homogeneização, centrifugue o homogeneizado (16.000 × g, 4 ° C) e transferir cuidadosamente o sobrenadante para um novo tubo de 1,5 mL com uma pipeta de 100 µ l.</li>
	<li>Adicionar o carregamento do buffer solução (5x) à proteína sobrenadante na proporção de 1:4 e desnaturar a proteína a 100 ° C por 5 min.</li>
	<li>Os níveis de expressão de proteínas no coração e outros tecidos de órgãos são mais monitorados pela análise ocidental do borrão (representante resultados são mostrados na Figura 2 e Figura 3) de acordo com o protocolo descrito anteriormente12.</li>
</ol>

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O presente relatório demonstra uma técnica modificada para injeção Intramiocárdica de vetores virais para manipulação do gene cardíaca, que foi modificado de um método para indução do infarto do miocárdio por Gao et al 13 atualmente, na vivo caracterização das funções do gene específico geralmente envolvem a geração de nocaute ou ratos transgénicos3,14,15,<...

Doença cardíaca é a principal causa de morbilidade e mortalidade em todo o mundo1,2. A falta de estratégias terapêuticas eficazes para doenças cardíacas fatais e incluindo infarto do miocárdio e insuficiência cardíaca atrai a exploração intensiva de pathomechanisms subjacentes e identificação de opções terapêuticas novela3. Para estas explorações científicas, manipulação genética cardíaco-específicas é amplamente utilizado4,5. Manipulação genética cardíaca pode ser conseguida editando genoma usando a nuclease efetoras como ativador de transcrição poderoso (TALEN) e agrupados regularmente intercaladas curtas palíndromos repetições (CRISPR) / proteína associada CRISPR 9 (Cas9) ferramentas, ou por entrega de materiais genéticos de gravidez ectópica (por exemplo, vetores de vírus, carregando genes que codificam proteínas de interesse)6. Embora a edição do genoma permite modificações precisas e spatiotemporal genoma em ratos vivos, ainda é uma prática demorada e trabalhosa,6. Alternativamente, manipulação de gene específico cardíaco pelo vírus vetor ou interferindo pequeno entrega complexa de RNA (siRNA) são rotineiramente realizada6.
Entrega de vetor de vírus para o coração de rato adulto é alcançada por aproximadamente duas estratégias: injeção sistêmica ou local. Injeção sistêmica de sorotipo cardiotropic de AAVs como AAV9 é não-invasiva para o gene específico cardíaca manipulação7. No entanto, esse método requer uma quantidade relativamente alta de vetor necessário para transdução eficiente e expressão gênica e pode resultar em significativa transdução do nontarget órgãos tais como o músculo e fígado7. Injeção local de vírus é conseguida por injeção Intramiocárdica ou entrega Intracoronário7. Intracoronário entrega leva a uma distribuição mais uniforme do vírus dentro do coração em comparação com injeção Intramiocárdica. No entanto, as desvantagens desta técnica são a lavagem rápida fora de vetores virais para a circulação sistêmica e transdução em órgãos nontarget8e sua exigência de dispositivos para medição de pressão durante a operação. Por outro lado, permite de injeção Intramiocárdica melhor retenção de vírus no miocárdio, bem como entrega de local específico, mas não consegue distribuir uniformemente viral vector7. Para pequenos animais, entrega Intracoronário é tecnicamente difícil de executar, enquanto sistêmica AAV9 injeção e injeção Intramiocárdica são mais comumente praticada4,5,7. Embora injeção sistêmica é fácil de realizar, injeção Intramiocárdica convencional requer toracotomia e ventilação mecânica, provoca dano tecidual extensa e é demorada.
Neste relatório, nós descrevemos um método fácil, economia de tempo e altamente eficiente para injeção Intramiocárdica. Adenovírus codificação luciferase e VDR ou shRNA alvo VDR, foi injetado para manipular a expressão de gene cardíaca. Uma vez dominado, esse método pode ser usado para manipulação genética, bem como a injeção de células ou outros materiais no coração do mouse.

<table border="0" cellpadding="0" cellspacing="0" width="914">
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			<td colspan="4" height="40" style="height:40px;width:916px;">Equipments</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Laminar flow sterile hood</td>
			<td style="width:267px;">Fengshi Animal Experimental&#160; Equipment Techonology Co., Ltd. (Soochow, China)</td>
			<td style="width:143px;">FS-CJ-2F</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Centrifuge</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">75005282</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Tissue grinding machine</td>
			<td style="width:267px;">Scientz Biotechnology Co., Ltd. (Ningbo, China)</td>
			<td style="width:143px;">Scientz-48</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">High temperature/high pressure sterilizer</td>
			<td style="width:267px;">Hirayama (Saitama, Japan)</td>
			<td style="width:143px;">HVE-50</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Isoflurane vaporizer&#160;</td>
			<td style="width:267px;">Matrix (Orchard Park, USA)</td>
			<td style="width:143px;">VIP3000</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">IVIS&#160; Lumina III imaging system</td>
			<td style="width:267px;">PerkinElmer (Waltham, USA)</td>
			<td style="width:143px;">CLS136334</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Precision&#160;balance</td>
			<td style="width:267px;">Sartorius (G&#246;ttingen, Germany)</td>
			<td style="width:143px;">28091873</td>
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			<td height="40" style="height:40px;width:351px;">Eppendorf pipette (100 &#181;L)</td>
			<td style="width:267px;">Eppendorf (Westbury, USA)</td>
			<td style="width:143px;">&#160;4920000059</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Eppendorf pipette (10 &#181;L)</td>
			<td style="width:267px;">Eppendorf (Westbury, USA)</td>
			<td style="width:143px;">&#160;4920000113</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Forceps</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp.&#160;</td>
			<td style="width:143px;">JD4020</td>
			<td style="width:156px;">Curved tip</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Hamilton syringe</td>
			<td style="width:267px;">Hamilton (Nevada, USA)</td>
			<td style="width:143px;">80501</td>
			<td style="width:156px;">Volume 50 &#956;L</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Micro-mosquito hemostat</td>
			<td style="width:267px;">F.S.T (Foster City, USA)</td>
			<td style="width:143px;">13011-12</td>
			<td style="width:156px;">Curved, tip width 1.3mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Needle holder&#160;</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China)</td>
			<td style="width:143px;">J32110</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Surgical scissors</td>
			<td style="width:267px;">F.S.T (Foster City, USA)</td>
			<td style="width:143px;">14002-12</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">1-mL Syringe</td>
			<td style="width:267px;">WeiGao Group Medical Polymer Co.,Ltd. (ShangDong, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Materials and reagents</td>
			<td style="width:267px;"></td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-GAPDH antibody</td>
			<td style="width:267px;">CST (Danvers,&#160; USA)</td>
			<td style="width:143px;">#2118</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-Luciferase antibody</td>
			<td style="width:267px;">Abcam (Cambridge, UK)</td>
			<td style="width:143px;">ab187340</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-rabbit IgG</td>
			<td style="width:267px;">CST (Danvers,&#160; USA)</td>
			<td style="width:143px;">&#160;#7074</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-VDR antibody</td>
			<td style="width:267px;">Abcam (Cambridge, UK)</td>
			<td style="width:143px;">&#160;ab109234</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Buprenorphine</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">PA175056</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Chloralic hydras</td>
			<td style="width:267px;">LingFeng Chemical (ShangHai, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Cryogenic Vials</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">375418</td>
			<td style="width:156px;">1.8 mL&#160;</td>
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			<td height="40" style="height:40px;width:351px;">Depilatory cream</td>
			<td style="width:267px;">Veet (Shanghai, China)</td>
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			<td style="width:156px;"></td>
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			<td height="40" style="height:40px;width:351px;">Dulbecco&#39;s phosphate buffered saline&#160;</td>
			<td style="width:267px;">Gibco (Grand Island,&#160; USA)</td>
			<td style="width:143px;">14040133</td>
			<td style="width:156px;"></td>
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			<td height="40" style="height:40px;width:351px;">Entoiodine</td>
			<td style="width:267px;">LiKang (Shanghai, China)</td>
			<td style="width:143px;">310132</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">EP tube</td>
			<td style="width:267px;">Sarstedt (Newton, USA)</td>
			<td style="width:143px;">PCR001</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Filter</td>
			<td style="width:267px;">Millipore (Bedford, USA)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">Pore size 0.2 &#181;m&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Isoflurane</td>
			<td style="width:267px;">Yipin Pharmaceutical Company (Hebei, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Luciferin</td>
			<td style="width:267px;">Promega (Madison, USA)</td>
			<td style="width:143px;">P1041</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Lysis buffer for western&#160; blot</td>
			<td style="width:267px;">Beyotime (Shanghai, China)</td>
			<td style="width:143px;">P0013J</td>
			<td style="width:156px;">Without inhibitors</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Ophthalmic cream</td>
			<td style="width:267px;">Apex Laboratories ( Melbourne, Australia))</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">PBS</td>
			<td style="width:267px;">Gibco (Grand Island,&#160; USA)</td>
			<td style="width:143px;">10010023</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Protease inhibitor cocktail</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">78438</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">5-0 silk suture</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Steel ball</td>
			<td style="width:267px;">Scientz Biotechnology Co., Ltd. (Ningbo, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">Width 1.5 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Syringe needle</td>
			<td style="width:267px;">Kindly Medical Devices Co., Ltd. (Zhejiang, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">30 gauge&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Warm mat</td>
			<td style="width:267px;">Warmtact Electrical Heating Technology Co., Ltd. (Guangdong, China )</td>
			<td style="width:143px;">NF-GNCW</td>
			<td style="width:156px;"></td>
		</tr>
	</tbody>
</table>

a simple and efficient method for in vivo cardiac-specific gene manipulation by intramyocardial injection in mice

Manipulação genética, especificamente no coração significativamente potenciar a investigação de doença cardíaca pathomechanisms e seu potencial terapêutico. Na vivo entrega cardíaco-específicas do gene é comumente conseguida entrega local ou sistêmica. Injeção sistêmica através da veia da cauda é fácil e eficiente em manipular a expressão de gene cardíaco usando vírus adeno-associado recombinante 9 (AAV9). No entanto, esse método requer uma quantidade relativamente alta de vetor para transdução eficiente e pode resultar na transdução de gene nontarget órgão. Aqui, descrevemos um método simples, eficiente e economia de tempo de injeção Intramiocárdica para na vivo manipulação gene específico cardíaca em ratos. Sob anestesia (sem ventilação), os músculos peitorais maiores e menores foram dissecados sem rodeios, e o coração de rato foi rapidamente exposto pelo manual exteriorização através de uma pequena incisão no quarto espaço intercostal. Posteriormente, adenovírus, codificação do luciferase (Luc) e receptor de vitamina D (VDR) ou RNA de gancho de cabelo curto (shRNA) segmentação VDR, foi injetado com uma seringa Hamilton o miocárdio. Subsequentes na vivo imagem demonstrou que luciferase foi com êxito Blumental especificamente no coração. Além disso, a análise ocidental do borrão confirmou o sucesso superexpressão ou silenciamento de VDR, no coração do mouse. Uma vez dominado, esta técnica pode ser usada para manipulação genética, bem como a injeção de células ou outros materiais, tais como nanogels no coração do mouse.

O protocolo do experimento e algumas das principais etapas para o método relatado são mostradas na Figura 1. Em 5 dias após a injeção Intramiocárdica de luciferase codificação de adenovírus (Adv-luc), na vivo por imagens em ratos injetado adv-luc indicado robusto superexpressão de luciferase especificamente no coração (Figura 2A, B), que foi confirmado por análise ocidental do borrão (<stro...

Watch this Scientific Journal Video about A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice at JoVE.com

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Aqui nós apresentamos um protocolo para a manipulação do gene específico cardíaca em ratos. Sob anestesia, os corações de rato foram exteriorizados através do quarto espaço intercostal. Posteriormente, adenovírus codificação de genes específicos foram injetados com uma seringa o miocárdio, seguido por medição de expressão de proteínas via na vivo de imagem e Western blot análise.

Um método simples e eficiente para In Vivo cardíaco-específicas do Gene manipulação por injeção Intramiocárdica em ratos

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

a-simple-efficient-method-for-vivo-cardiac-specific-gene-manipulation

Research

JoVE Journal

Biology

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

15.7K visualizações.  The First Affiliated Hospital of Bengbu Medical College. Aqui nós apresentamos um protocolo para a manipulação do gene específico cardíaca em ratos. Sob anestesia, os corações de rato foram exteriorizados através do quarto espaço intercostal. Posteriormente, adenovírus codificação de genes específicos foram injetados com uma seringa o miocárdio, seguido por medição de expressão de proteínas via na vivo de imagem e Western blot análise.

Vídeo: A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Um método simples para genotipagem Accurate

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Biologia

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usando Seqüência de Detenção SECM como uma ferramenta para isolar Ribossomos Polipeptídeos encadernados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Técnica simples e eficiente para a preparação de suspensões de células testiculares

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

In Vivo SiRNA transfecção e Gene Knockdown em Spinal Cord via rápida não invasiva lombar intratecal Injeções em Ratos

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente Geração induzida pelo homem células-tronco pluripotentes a partir de células somáticas humanas com Sendai-vírus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Injeções intravenosas em Neonatal Ratos

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Isolamento de filtração de Ácidos Nucleicos: Um Método de extracção de DNA simples e rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.